Open Journal of Soil Science, 2012, 9, 312-319 Published Online September 2012 (
Analysis of the Bacterial Communities in Lime Concretion
Black Soil upon the Incorporation of Crop Residues
Shao-Qiang Tao, Qiang Xia, Lin Zhu, Jing-Jing Chen, Ya-Nan Wang, Bing Qin
College of Resources and Environmental Sciences, Anhui Agricultural University, Hefei, China.
Received June 15th, 2012; revised July 20th, 2012; accepted July 31st, 2012
To analyze the bacterial communities in lime concretion black soil upon the incorporation of crop residues for two years
in wheat-maize system, total DNA was directly extracted and PCR-amplified with the F357GC and R518 primers tar-
geting the 16S rRNA genes of V3 region. The amplified fragments were analyzed by perpendicular DGGE. Analyzing
of species richness index S and Shannon diversity index H revealed that there was a high diversity of soil bacterial
community compositions among all treatments after incorporation of crop residues and fertilizing under field conditions.
Eleven DGGE bands recovered were re-amplified, sequenced. Phylogenetic analysis of the representative DGGE fin-
gerprints identified four groups of the prokaryotic communities in the soil by returning wheat residues and fertilizing
under field conditions. The bacterial communities belonged to gamma proteobacterium, Cupriavidus sp, halophilic
eubacterium, Acidobacterium sp, Sorangium sp, delta proteobacterium, Streptococcus sp and Streptococcus agalactiae
were main bacterial communities. Principal Component Analysis (PCA) showed that there were the differences in DNA
proles among the six treatments. It showed that wheat residue returning, maize residue returning and fertilizing all can
improve bacterial diversity in varying degrees. As far as improvement of bacterial diversity was concerned, wheat resi-
due returning was higher than fertilizing, and fertilizing higher than maize residue returning.
Keywords: Crop Residues; Bacterial Community; Lime Concretion Black Soil; Denaturing Gradient Gel
Electrophoresis (DGGE); 16S rDNA; Wheat-Maize System
1. Introduction
Plant residues are an important source of nutrients in
both natural and agricultural ecosystems, where syn-
chronous plant growth and residue decomposition are
essential for soil fertility. Fresh plant material (e.g. litter)
represents a readily available substrate for both soil fauna
and soil microorganisms. The main mineralization acti-
vity is performed by soil microbial communities, and the
composition of the organic residues controls the decom-
position rate and the related release of nutrients [1].
It has been reported that only a small fraction of mi-
croorganisms in nature are culturable. Therefore, to study
the total microbial communities involved in organic ma-
tter decomposition, culture-independent and culture-de-
pendent are needed.
Molecular techniques offer new opportunities for
analysis of the structure of a microbial community [2]. In
particular, sequence variation in rRNA genes has been
exploited for inferring phylogenetic relation-ships among
microorganisms [3] and may be used to estimate the ge-
netic diversity of complex microbial communities in na-
tural ecosystems [4-7]. All prokaryotes have 16S rRNA
genes whose average length is about 1500 bp. There are
both conserved and variable regions (the V1-V9 regions),
and sufcient information has been compiled with which
to conduct for reliable phylogenetic analyses [8,9]. De-
naturing gradient gel electrophoresis (DGGE) allows one
to directly determine the richness and evenness of the
dominant microbial species using 16S rRNA gene am-
plicons and, thus, to prole the corresponding microbial
populations in both a qualitative way and a semiquantita-
tive way [10-13]. The diversity can be estimated from the
number of 16SrRNA gene sequence similarity groups (i.e.
the number of DNA bands on the DGGE gel [4] and the
intensity of DGGE bands. Each band is assumed to rep-
resent an operational taxonomic unit, which is called a
species for simplicity [14].
It is important for the typical wheat-maize cropping
system of two crops a year in the Huaibei Plain (HP) to
the food security of Anhui, China. The crop residues
produced by the systems used to burn and bring about
environment pollution, which seriously affect the sus-
tainable development of agriculture. A field experiment
was conducted to study the diversity of soil bacteria
Copyright © 2012 SciRes. OJSS
Analysis of the Bacterial Communities in Lime Concretion Black Soil upon the Incorporation of Crop Residues 313
communities in the Wheat-maize system under incorpo-
ration of crop residues.
2. Materials and Method
2.1. Experimental Site and Soil
A eld experiment was conducted in rice-wheat cropping
systems for two years (2008-2010) in Mengcheng, Anhui,
China. The climate is semi-tropical monsoon with hot
and rainy summers and cold winters. The area receives
850 mm annual rainfall, about 80% of which occurs from
June to September. The mean annual temperature is be-
tween 14.3˚C to 16.7˚C.
The alluvial soil of experimental site was a typical
lime concretion black soil, well drained with pH 6.5 and
silty clay loam in texture. The soil had soil organic mat-
ter 1.25%, and available N, P and K 80.2, 15.4 and 100.3
mg·kg1, respectively. Soils were collected from the top
layer (0 - 20 cm) of the field at mid filling stage of maiz e.
The soil was homogenized by sieving (2-mm mesh), and
stored at 4˚C before use.
2.2. Treatments and Crop Management
Treatments were arranged according to a completely
randomized design with three replicates (Table 1).
Take the quantity of corn straw returning by grinding
and burying. The amount of maize residue was about
12,000 kg·ha1. Wheat straw was returned to the field by
grinding and bestrowing. The amount of wheat residue
was about 7500 kg·ha1. Wheat (cultivars Yan Nong 19)
was transplanted in the plots at row spacing of 20 cm.
Planting density reach to 2.7 × 106 ha1 individual plant,
Maize (cultivars Zheng Dan 958 ) at row spacing of 60
cm and planting density reach to 6.75 × 104 ha1. During
wheat growth period, Nitrogen as urea was applied at
240 kg·N·ha1, base fertilizer ratio to additional fertilizer
was 5.5:4.5, combined with landpreparation 600 kg·ha1
compound fertilizer and 240 kg·N·ha1 were applied in
related plot. Other 45% Nitrogen as additional fertilizer
was applied in shooting period of wheat. During maize
growth period, Nitrogen as urea was applied at 300
kg·N·ha1, 450 kg·ha1 compound fertilizer (N:P2O5:K2O
= 15:15:15) as base fertilizer was applied, 112.5 and 120
kg·N·ha1 were applied respectively at six and twelve
leaves expanded.
2.3. DNA Extraction of the Soil
Total community DNA extraction of the soil was carried
out by the method of SDS-based DNA extraction as de-
scribed by Zhou et al. (1996) with some modification
[15]. Extraction buffer for removing humic acids (5 ml of
100 mmol·L1 Tris-HCl, 100 mmol/L Na4P2O7, 100
mmol·L1 sodium EDTA, 100 mmol·L1 NaCl, 4% de-
Table 1. The field trial design of wheat and maize residue
Number Treatment
CK No wheat and maize residue returning, No fertilizing
CK-F No wheat and maize residue returning, Fertilizing
M-F Maize residue returning and no wheat residue returning,
WM-NF Wheat and maize residue returning, No fertilizing
WM-F Wheat and maize residue returning, Fertilizing
W-F Wheat residue returning and no maize residue returning,
grease milk powder, pH 10.0) was added with 0.5 g (wet
weight) of soil, and mixed by vortexing for 3 min. The
mixture was incubated at 65˚C for 10 min in a water-bath,
with intermittent vortexing. The mixture was centrifuged
at 3000 g for 5 min. The supernatant was discarded and
pellet was washed with the removing humic acids extrac-
tion buffer once more up to the color of the supernatant
almost same as the buffer. The pellet was mixed with
0.54 ml DNA extraction buffer (100 mmol·L1 Tris-HCl,
100 mmol·L1 Na3PO4, 100 mmol·L1 sodium EDTA,
1.5 mol·L1 NaCl, 1% CTAB, pH 8.0) and added with 20
µl proteinase K (10 mg·mL1). The mixture was shaked
at 225 rpm·min1 for 0.5 h at 37˚C in table concentrator.
Then, 60 µl 20% SDS was added to the mixture, incu-
bated at 65˚C for 2 h in a water-bath, with intermittent
gently mixing. The sample was centrifuged at 6000 g for
10 min at room temperature. The supernatant was col-
lected and the soil pellet re-extracted with further extrac-
tion buffer (90 µl), incubated at 65˚C for 10 min and
centrifuged as above. The supernatant uid was trans-
ferred to centrifuge tubes (2 ml) and added with a equal-
volume of phenol:chloroform:Isoamyl alcohol (25:24:1,
v/v/v), mixed, freezing in ice-bath for 10 min. Mixtures
were centrifuge at 16,000 g for 10 min at 4˚C. The aque-
ous phase was extracted with hydroxybenzene:chloro-
form:Isoamyl alcohol two times. After then, the upper
aqueous phase was added equal volume of hydroxyben-
zene:chloroform:Isoamyl alcohol (25:24:1, v/v/v), cen-
trifuging at 16,000 g for 10 min at 4˚C. The partially
puried nucleic acid pellet resuspended in 20 mL of TE
(10 mmol·L1 Tris-HCl, 1 mmol·L1 sodium EDTA, pH
8.0). Potassium acetate (7·5 mol·L1) was added to a
nal concentration of 0·5 mol·L1. The sample was trans-
ferred to ice for 5 min, then centrifuged (16,000 g, 30
min) at 4˚C to precipitate proteins and polysaccharides,
and DNA was precipitated by adding 0·6 volume iso-
propanol. After 1 h at room temperature, DNA was pre-
cipitated by centrifugation (16,000 g for 30 min) and
resuspended in TE (20 µL).
2.4. PCR
The total DNA preparation of each sample was amplied
Copyright © 2012 SciRes. OJSS
Analysis of the Bacterial Communities in Lime Concretion Black Soil upon the Incorporation of Crop Residues
by PCR with a 2720 Thermal Cycler (Applied Bio-sys-
tems, USA). The variable V3 region of 16S rRNA gene
sequences from nucleotide 8 to nucleotide 1512 (Es-
cherichia coli numbering) was amplied by PCR by us-
ing eubacterial primers 27 F (5’-AGAGTTTGATCC-
TGGCTCAG-3’ ) and 1492 R (5’-GGTTACCTTGTT-
ACGACTT-3’) at first, and then nested PCR was per-
formed by using primers F357(5’-GC-CCTACGGG-
AGGCAGCAG-3’) together with a GC clamp (5’
g-3’) and R518 (5’-ATT ACC GCG GCT GCT GG-3’)
and the hot-start touchdown protocol described by Muy-
zer et al [2]. DNA extracted from the soil was amplied
with a PCR mixture (25 μl) containing 2.5 μl of Mg-
containing 10 × buffer, 2 μl of a 2.5 mmol·L1 de-oxynu-
cleoside triphosphate mixture, 5 pmol of forward and
reverse primers respectively, 1 μl of the DNA solution
(1:10 dilution of DNA extraction), and 0.75 μl of Ex-
pand High Fidelity DNA polymerase ((TaKaRa). The
polymerase was added after a hot-start procedure (5 min
at 94˚C, followed by 5 min at 80˚C). PCR was performed
with a 2720 Thermal Cycler by using the following pro-
tocol: 1 min at 94˚C (denaturation), 45 s at 65˚C (anneal-
ing,), and 3 min at 72˚C (elongation) with a 0.5˚C touch-
down every cycle during annealing for 20 cycles, fol-
lowed by 10 cycles with an annealing temperature of
55˚C and a nal cycle consisting of 10 min at 72˚C.
The absorbances of the DNA at wavelengths of 230,
260, 280 and 320 nm were determined with a spectro-
photometer (NanoDrop® ND-1000, Thermo, USA) and
1% agarose gels were run to visualize the integrity of the
2.5. DGGE
DGGE was performed using DcodeTM Universal Muta-
tion Detection System (Bio-Rad, Hercules, CA, USA).
For each sample, 400 ng of PCR product was loaded onto
a poly-acrylamide gel (8%, wt/vol) with a 45% to 65%
denaturant gradient, A denaturing gradient consisting of
100% denaturant is defined as 7 M urea with 40 % (v/v)
Electrophoresis was carried out at 100 v for 16 h (with an
initial 30 min at 40 v ) at 60˚C in 0.5 × TAE buffer (4.84
g of Tris base per liter, 11.42 ml of acetic acid per liter,
20 ml of 0.5 M EDTA per liter; pH 8.0). Gels were
stained in 1:10,000 (v/v) SYBR® Green for 30 min and
visualized under UV irradiance. Gel images were cap-
tured with a Gel Doc-ItTM documentation system. DGGE
bands migrating to the same position in different lanes in
the gel were considered to have the same sequence. The
scanned gels containing DNA band proles were ana-
lyzed to determine the intensity of each band using
Quantity One 4.6 software (Bio-Rad, Hercules, CA,
USA). Selected bands were cut from DGGE gels and
transferred to 1.5 ml micro tubes and purified by using
Agarose Gel DNA purification Kit (TaKaRa), and were
sub cloned into the pMD18-T vector, then transformed
into E. coli strain DH5α and sequenced.
2.6. Data Analyses
DNA sequence analyses were edited and reformatted
using the ChromasPro software. A similarity search for
the nucleotide sequences of 16S rDNA genes of the test
isolates was carried out online at using the BLAST search
program for the nucleotide database maintained in Gen-
Bank. Finally, the test sequences were compared with
those of selected related species and the phylogenetic
relationship of these sequences was determined by
MEGA4 software. Bacterial diversity in the samples was
estimated in two different ways: as S, species richness
(the number of DGGE fragments detected disregarding
their relative intensities), and H, the Shannon index of
bacterial diversity. The Shannon diversity index was
calculated as (H) = −ΣPilnPi = −Σ(Ni/N)ln(Ni/N) based
on the relative band intensities, as formulated by Eichner
et al. [16]. Pi was dened as ni/N, where Ni is the area of
a peak in intensity and N the sum of all peak areas in the
lane proles.
To estimate the diversity of bacterial communities in
lime concretion black soil after the incorporation of crop
residues in wheat-maize system, the data obtained from
the DGGE patterns, based on band intensity and position,
were analyzed by principal component analysis. Principal
component analysis was performed using SPSS (version
3. Results
3.1. DNA Extraction from Soil Samples and
Specic PCR Amplication of 16S rRNA
DNA extracted from each soil sample of 6 treatments has
much high integrality. The DNA contents of the crude
extracts were measured. The A260/A230 absorbance ratio
indicates polysaccharide or polyphenolic contamination,
and the A260/A280 ratio indicates protein contamination
[17]. The A260/A280 ratios of DNA extraction from soil
samples were all about 1.4, but the A260/A230 ratios were
all about 0.7. It suggested that humic acid had yet not
been wiped out fully from the DNA extraction The target
gene for the bacterial communities could successfully be
amplied in all extracts Using primers 27 F and 1492 R,
a special DNA fragment amplied by first round PCR
was gained in each soil samples The length of the special
DNA fragment was about 1533 bp. At second round PCR
Using primers F357 and R518, a special DNA fragment
(234 bp) was obtained in each soil samples by diluting
the product of the first round PCR 1:100 as template.
Copyright © 2012 SciRes. OJSS
Analysis of the Bacterial Communities in Lime Concretion Black Soil upon the Incorporation of Crop Residues 315
Equal quantities of amplied DNA (234 bp) were loaded
into the slots of the different lanes on the gel for com-
parison of the diversity of soil bacteria in different treat-
ment by DGGE.
3.2. DGGE
The banding profile of 16S rDNA DGGE analysis re-
vealed that the populations of bacteria varied among all
soils of different treatments (Figure 1).
The different DNA fragments were the same length
but had different base sequences (operational taxonomic
units or species). Between 28 and 38 bands were distin-
guished in the soils of all treatments (Table 2 ). The low-
est number of bands was in the control treatment CK
with no wheat and maize residue returning, no fertilizing,
and the highest number was obtained from the treatment
W-F with wheat residue returning and fertilizing, but no
maize residue returning. Dominant bands were present
particularly WM-NF, WM-NF and W-F, in contrast to
CK, CK-F, M-F, where the bands were more similar and
had higher intensity. Disregarding the relative intensities
of detected fragments, S (species richness) among WM-
NF, WM-NF and W-F were higher than CK, CK-F and
M-F. It indicated that band number depended on wheat
residue returning, in addition, fertilizing can also add the
band number in some degree. Considering to the soil
samples we got at maize growth period, it was wheat
residues but maize residues that had more effects on the
bacterial diversity and quantity. Shannon index H of CK
treatment was the lowest as 2.69, where no crop residue
returning and no fertilizing, and the one of CK-F, WM-
NF, WM-NF and W-F treatment was higher than CK
treatment. The index H of CK-F treatment was the high-
est to 3.06 (Figure 2).
Figure 1. DGGE patterns of 16S rRNA gene sequences of
soil samples in lime concretion black soil after the incorpo-
ration of crop residues in wheat-maize system.
soil bacteria Shannon index
Figure 2. Soil bacteria communities of different species di-
versity index.
Table 2. Number of DGGE bands in DGGE profiles of bac-
Bands 28 29 31 35 36 38
Since we were just considering the technique to de-
scribe the genetic diversity associated with the effect of
wheat residues inoculation to soil and fertilizing under
field conditions, we decided to use the most representa-
tive banding profile to construct phylogenetic relation-
ship. In lane W-F of Figure 1, eleven selected bands
were cut from DGGE gels and sequenced. The phyloge-
netic relationship of these sequences was showed as Fig-
ure 3. Using the program BLAST, sequences with most
similarity to reference strains were found in the GenBank
database. The result showed that the bacterial communi-
ties belonged to Cupriavidus sp, gamma proteobacterium,
Streptococcus sp, Sorangium sp, Acidobacterium sp,
delta proteobacterium during the incorporation of crop
residues in wheat-maize system, thereinto, Streptococcus
sp and Acidobacterium sp were main bacterial communi-
Principal Component Analysis (PCA) showed that
there were the (qualitative) differences in DNA proles
among the six treatments (Figure 4). The crop residue
returning and fertilizing showed divergent development
of bacterial communities which was associated with dif-
ferent diversity indices. PC1 contributed much more than
PC2 to bacterial community. PC1 and PC2 constitute
63.15% of total variation. There were much similarity
between CK-F and W-F treatment. Though there was no
incorporation of crop residue in CK-F treatment, it had
been applied by fertilizer. This indicates that bacterial
communities developed in the process of crop litter re-
turning or fertilizing.
4. Discussion
Denaturing gradient gel electrophoresis (DGGE) is a gel
electrophoresis method used to separate DNA fragments
Copyright © 2012 SciRes. OJSS
Analysis of the Bacterial Communities in Lime Concretion Black Soil upon the Incorporation of Crop Residues
Copyright © 2012 SciRes. OJSS
FJ561528. 1| Uncul tured bac terium clo(2)
FJ468390. 1| Uncul t ured bac terium clo. . .
FJ561528. 1| Uncul t ured bac terium clo. . .
JF429008. 1| Uncul t ured bact erium clo. ..
EF 663985. 1| Unc ul t ured gam m a prot eoba. . .
EU790328. 1| Unc ul t ured bacteri um c l o. ..
GU566329. 1| Cupriavidus sp. A P1(2010)...
FN994942. 1|Unc ultured hal ophi l i c euba. . .
JF835730. 1|Unc ul tured bacteri um clone...
FR716177.1| Unc ul tured bacteri um par. ..
FR732350.1| Unc ul tured soil bacteri u...
FN870288.1| Unc ul tured bacteri um part ...
HQ995661.1| A ci dobacteriac eae bacter...
JF904151. 1| Unc ul tured bact erium clo...
HQ730663.1| Unc ul t ured A ci dobacterium...
E F072379. 1| Unc ul t ured A ci dobacterial...
DQ830568. 1| Unc ul tured bact erium clo...
DQ830170. 1| Unc ul tured bact erium clo...
FN567901. 1| Unc ultured bact eri um par. ..
GQ376983. 1| Unc ul t ured bac teri um c l o...
HQ018194. 1| Unc ul tured Sorangi um sp. ...
E F 663798. 1| Uncul tured delta proteoba...
E F 072005. 1| Uncul tured delta proteob...
DQ830380. 1| Uncul t ured bac terium clo. . .
GU132048.2| Unc ul t ured S t reptococcus . . .
HQ536043.1| Unc ul t ured bac teri um c l o...
HQ536039.1| Unc ul t ured bac teri um c l o...
GU132035.2| Unc ul t ured S t reptococcus . . .
JF423948. 1| S treptococ cus agalact i ae...
GU146033.2| Unc ul t ured S t reptococcus . . .
GU146032.2| Unc ul t ured S t reptococcus . . .
Figure 3. Dendrogram of phylogenetic relationship based on the denaturing gradient gel electrophoresis (DGGE) schematic
banding profiles.
of the same length, but containing different base-pair
sequences; it is used to determine the presence and abun-
dance of different microbial species in a mixed popula-
tion [18]. It was put forward first by Fischer and Lerman
to detect single base substitutions within long DNA se-
quences [19]. Muzyer et al. apply this approach to ana-
lyzing the genetic diversity of complex microbial popu-
lations, which is based on the separation of polymerase
chain reaction-amplified fragments of genes coding for
16S rRNA ribosomes, all the same length, by DGGE [6].
DGGE exploits base-pair sequence difference to separate
amplified DNA on a high-resolution polyacrylamide gel
along a denaturing gradient. Sequences differing in base
composition denature at different locations on this gel.
The number of bands on the gel is therefore indicative of
the gene diversity in the original sample, such as a DNA
Analysis of the Bacterial Communities in Lime Concretion Black Soil upon the Incorporation of Crop Residues 317
Figure 4. Qualitative PCA of 16S rRNA gene denaturing
gradient gel electrophoresis (DGGE) profiles for bacterial
extract from soil.
Crop residues have complex structure and are combi-
nations of many different ingredients such as hemicellu-
lose, cellulose, lignin and water-soluble polysaccharide.
The degradation of these substances in the soil demand
associative action of multi-microorganisms. Kimura’s
study showed that there existed complicated microflora
in the process of stalk decomposition [20]. Only an esti-
mated 0.1% - 1% of the naturally occurring bacteria have
been isolated and characterized so far [21]. Selective
enrichment cultures fail to mimic the conditions that par-
ticular microorganisms require for proliferation in their
natural habitat. Furthermore, many microorganisms are
bound to sediment particles and are thus not detected by
conventional microscopy [2].
Molecular biological techniques offer new approach
for the analysis of the structure and species composition
of microbial communities. In particular, sequence varia-
tion rRNA has been exploited for inferring phylogenetic
relationships among microorganisms [3]. These tech-
niques have also been applied to determining the genetic
diversity of microbial communities and to identifying
several uncultured microorganisms [22,23]. They consti-
tute the cloning of ribosomal copy DNA or polymerase
chain reaction (PCR amplified ribosomal DNA (rDNA)
followed by sequence analysis of the resulting clones. By
using 16S rDNA in this community DNA for analysis,
similarly it is possible to reveal the soil microbial com-
munity after the incorporation of crop residues in wheat-
maize system.
Extraction of DNA from soils always results in coex-
traction of humic substances which interfere with DNA
detection and measurement. This contamination can in-
hibit Taq DNA polymerase in PCR interfere with restric-
tion enzyme digestion [24,25], and reduce transformation
efficiency [26] and DNA hybridization specificity [27].
Since humic substances are difficult to remove, DNA
purification is a critical step following direct extraction
to obtain DNA of sufficient purity. Zhou et al. indicated
that both CTAB and PVPP can effectively remove humic
materials [15]. In our study, we had also made use of the
de-humic effect of sodium pyrophos-phate and degrease
milk powder for removing humic acids, besides adding
CTAB and PVPP. Finally, the bacterial variable V3 re-
gion of 16S rDNA was amplied by diluted template and
nested PCR This simple and practicable method can
eliminate inhibition of humic substances to Taq DNA
polymerase on the whole.
The DNA band pattern obtained by DGGE is an at-
tractive way to study complex communities in environ-
mental samples because most of the bacterial genotypes
(species) gave one band [11]. The intensities of the DNA
bands reected the relative levels of the bacterial strains.
Bacterial DNA proles obtained by DGGE can be used
as a semiquantitative measure of bacterial diversity, and
we therefore used this method to study bacterial diversity
after incorporation of crop residues and fertilizing under
field conditions in agricultural soils.
Analyzing of DGGE profiles reveal that either crop
residue returning to soil or fertilizing can increase spe-
cies richness index, S and the Shannon index of bacterial
diversity, H. Moreover, Shannon index H was consistent
with species richness index S as a whole among all treat-
ments (exception for CK-F treatment). The disappear-
ance of dominating bands and the subsequent develop-
ment of a more uniform band pattern of CK-F treatment
can be interpreted as follows: r strategists (opportunists)
that prevailed in soil of fertilizing were replaced by a
variety of K strategists (persisters).This resulted in a spe-
cial high H value of CK-F treatment.
The DNA band patterns obtained from amplified 16S
rDNA V3 region gene sequences and DGGE indicated
that the structure and diversity of bacterial communities
changed significantly by crop residue returning or fertile-
izing. The numbers of distinct DGGE bands for all
treatments were between 28 and 38. The lowest number
was in the control treatment CK.
The number of DGGE bands was taken as an indica-
tion of species in each sample. The relative intensity of
each DGGE band and the sum of all the surfaces for all
bands in a sample were used to estimate species abun-
dance [28,29]. The Shannon index of diversity (H) was
used to determine the complexity of the DGGE bands
from soils of different treatments. A higher diversity was
observed in the soil of CK-F treatment than others. By
using the Shannon index of diversity in combination with
the cluster analysis of the DGGE banding patterns based
on the similarity coefficient, we were able to monitor a
whole range of community responses from all the treat-
ment soils. Genetic diversity of soil samples was mea-
Copyright © 2012 SciRes. OJSS
Analysis of the Bacterial Communities in Lime Concretion Black Soil upon the Incorporation of Crop Residues
sured as bands on denaturing gradient gel electrophoresis
(DGGE) of amplified 16S rDNA sequences from the soil
community DNAs. When analysed by Shannon index
(H), the highest genetic biodiversity (H = 3.061) was
found in CK-F treatment with no wheat and maize resi-
due returning but fertilizing; the poorest biodiversity (H
= 2.692) in control treatment (CK) with no wheat and
maize residue returning, no fertilizing. The Shannon-
Weaver diversity index (H) was calculated from the
DGGE profiles for each treatment. High H indicates a
high diversity in the microbial community. In the present
study, considerable qualitative differences in the com-
munity structure were found in the six treatments. With
CK-F and W-F (wheat residue returning and no maize
residue returning, fertilizing) treatments, the bacterial
diversity was higher than with the other treatments, indi-
cating that the fertilizing and wheat residue returning
possibly enhanced the microbial community.
Simultaneously, the bacterial diversity of CK treat-
ment was lower than the one of other treatments accord-
ing to Shannon index of diversity. We can infer that the
bacterial diversity be closely related to the crop residue
returning and fertilizing which add nutriment for bacteria.
The number and intensity of bands on denaturing gradi-
ent gel electrophoresis among all treatments were diffe-
rent. It showed that the predominant bacteria in each
treatment were different.
Wheat residue returning, maize residue returning and
fertilizing all can improve bacterial diversity in varying
degrees. Contribution of PC1 was about 37.1%, and the
one of PC2 was about 26.1% (Figure 4). The result of
principal component analysis (PCA) showed that bacte-
rial community of CK and M-F treatment was different
from the one of the others. The difference of them lied
with that there was no wheat residue returning in CKand
M-F treatment. On the other hand, CK-F treatment had
the highest index H to 3.06 (Figure 2). It suggested that
fertilizing have much effect on bacterial community un-
der no crop residue returning. In conclusion, as far as
improvement of bacterial diversity is concerned, wheat
residue returning was higher than fertilizing, and fertiliz-
ing higher than maize residue returning.
The results of this study clearly demonstrate the im-
pacts of crop residue returning and fertilizing on the bac-
terial community composition of soils. The extensive
cloning and sequencing of PCR-DGGE bands proved to
be a powerful tool in assessing community structure dif-
ferences in soils. Our future objectives in studying the
impacts of management on microbial populations are
aimed at attempting to understand the relationships
which may exist between microbial community structure
and function.
In conclusion, microbial diversity increased during the
course of crop residue returning to soil or fertilizing. Not
only crop residue returning to soil but also fertilizing
affected the development of bacterial community. The
effect of interaction between crop residue returning to
soil and fertilizing on soil bacterial community need
making a further investigation.
5. Acknowledgements
This work was supported by grants From the National
Natural Science Foundation of China (Grant No. 2007-
BAD89B10) and Anhui Nature Science Foundation,
China (Grant No.090411026).
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